Effects Of MG-132 On Trans-differentiation And Apoptosis Induced By TGF-β₁ In Human Tubular Epithelial Cells

Objective:To study the effects of proteasome inhibitor MG-132 on trans-differentiation and apoptosis in HK-2 cells cultured in vitro and their molecular mechanism,and to approach the mechanism in preventing and treating tubular interstitial fibrosis in chronic kidney diseases.Methods and Results:Part 1:The effect of proteasome inhibitor MG-132 on transdifferentiation induced by TGF-β₁ in human tubular epithelial cellsMethods:Cultured HK-2 cells were divided into 4 groups:(1)control group;(2) TGF-β₁ group(5ug/L);(3)MG-132 group(2.5μM);(4) MG-132+TGF-β₁ group(the cells were pretreated by MG-132 2.5μM for 30rain,then added TGF-β₁ 5ug/L).After treated for 24h,the cells were collected.The expression of Smurf1,Smurf2 and Smad7 mRNA were tested by RT-PCR.The expression of Smurf1,Smurf2 and Smad7 protein were detected by Western Blot.The expression of FN andα-SMA in the HK-2 cytoplasm were traced by immunohischemistry.Results:The expression of Smurf1,Smurf2 mRNA in TGF-β₁ group increased and the Smad7 mRNA decreased in comparison with the control group(P＜0.05).The expression of Smurf1,Smurf2 protein in TGF-β₁ group increased while the Smad7 protein decreased in comparison with the control group(P＜0.05).The expression of FN andα-SMA in the HK-2 cytoplasm increased in TGF-β₁ group.The expression of Smurf1,Smurf2 mRNA in MG-132 group decreased significantly while the Smad7 mRNA decreased mildly in comparison with TGF-β₁ group(P＜0.05),the Smad7 protein also increased(P＜0.05).The expression of FN andα-SMA in MG-132 group decreased in comparison with TGF-β₁ group(P＜0.05).Part 2:The effect of proteasome inhibitor MG-132 on cell apoptosis of human tubular epithelial cellsMethods:Cultured HK-2 cells were divided into two groups:(1) control group;(2) MG-132 treated group(2 subgroups of low concentration group of 0.25～2.75uM and high concentration group of 3.0～5.0uM).The cells apoptosis rate were tested by flow cytometry.The cell morphologic changes were traced with inverted light microscope and fluorescence microscope after dyed by Hoechst 33258.The expression of p-IκB-αand IκB-αwere detected by Western Blot.Results:With the concentration of MG-132 increasing,the early apoptosis rate of HK-2 increased at the peak concentration of 2.5～3.0uM,then decreased gradually while the lately apoptosis rate and total death rate of HK-2 increased by S-like curve.The cell apoptosis morphology changed accordingly.The expression of p-IκB-αand IκB-αwere increased in the MG-132 treated group in comparison with the control group.Conclusion:MG-132 can inhibit the expression of Smurf1 and Smurf2 induced by TGF-β₁ and restore the expression of Smad7 in HK-2,down-regulate the expression of FN andα-SMA,which is mechanism of MG-132 preventing the cells from trans-differentiation;on the other hand,MG-132 can lead HK-2 cells to apoptosis through inhibiting the activity of NF-κB.